Transgenic Plants
It is known that new and altered traits (so-called “novel traits”) can be imparted to crop species by recombinant DNA technology. In order to derive these crops with novel traits, a method to insert recombinant DNA into the crop genome is required. This method, commonly referred to as transformation, is technically challenging and requires significant effort in developing the protocols for culture, transformation itself and regeneration of whole plants. In some species transformation has become routine, while in other species transformation remains difficult and time-consuming. Nevertheless, some crop varieties that were genetically engineered to express novel traits have been released into the commercial production chain, and others are undergoing field trials in preparation for commercial release
Many of these transgenic crop varieties have novel traits that provide altered phenotypes. These phenotypes include novel compositions, enhanced resistance to pests, disease or environmental stresses, and tolerance to herbicides. Such tolerance provides new means to control weeds and new production opportunities for farmers.
Many current novel traits in commerce affecting agronomic characteristics are collectively referred to as “input” traits, i.e. those traits that relate to the economics of production. For example, herbicide tolerance is an input trait as it allows farmers more options in controlling weeds; typically the costs of weed control can be lowered by these novel herbicide tolerances. Thus the economics of production or “inputs” required to grow the crop are favorably altered. Other traits such as resistance to insects can lower the costs for farmers through reduced chemical insecticide applications.
In addition to input traits, there are “output” traits that alter the composition or quality of the harvested plant. Such traits impact the final products or “outputs” from a crop and can include altered oil or meal composition, reduced antinutritional content and crops with altered processing characteristics. There has been a considerable effort towards the development of crops with output traits that provide new products, economic value and increased utility.
Some traits are classified as high-value “output” traits. Such traits reside in crop plants used for “molecular farming” to produce novel proteins with commercial or pharmaceutical applications. Molecular farming holds considerable promise for the economical production of large volumes of commercially useful and valuable proteins. Use of crop plants to mass produce proteins offers many advantages over fermentation technology including: ease of production; stability of the product when synthesized in plant storage organs such as tubers or seed; and possibility of recovering valuable co-products such as meal, oil or starch from the plants.
Proteins contemplated for mass production by molecular farming include industrial enzymes; for example, those derived from microbial sources such as proteases, starch or carbohydrate modifying enzymes (e.g. alpha amylase, glucose oxidase, cellulases, hemicellulases, xylanases, mannanases or pectinases). Additionally, the production of enzymes such as ligninases or peroxidases which are particularly valuable in the pulp and paper industry, has been suggested within various crop species. Other examples of commercially or industrially important enzymes which can be produced using molecular farming are phosphatases, oxidoreductases and phytases. The number of industrially valuable enzymes is large and plants offer a convenient vehicle for the mass production of these proteins at costs anticipated to be competitive with fermentation.
Additionally, molecular farming is being contemplated for use in the production and delivery of vaccines, antibodies (Hein, M. B. and Hiatt, A. C., U.S. Pat. No. 5,202,422), peptide hormones (Vandekerckhove, J. S., U.S. Pat. No. 5,487,991), blood factors and the like. It has been postulated that edible plants which have been engineered to produce selected therapeutic agents could provide a means for drug delivery which is cost effective and particularly suited for the administration of therapeutic agents in rural or under-developed countries. The plant material containing the therapeutic agents could be cultivated and incorporated into the diet (Lam, D. M., and Arntzen, C. J., U.S. Pat. No. 5,484,719).
In total, the novel input and output traits contemplated for crop plants are very broad in scope and can lead to the development of numerous new products and processes. Accordingly, reliable means to produce plants with novel traits and incorporate the initial transgenic plants into breeding and variety development programs are important tools for the delivery of these products into commerce.
A problem of transgenic plant production is that upon recovery of a transgenic event in a plant cell, a considerable effort is needed to recover morphologically normal, fertile plants for use in subsequent breeding schemes. Thus methods that allow for the simple identification of plants that have received the transgene is a primary objective for commercialization.
The selection or identification of transgenic plants by reliable methods that do not require biochemical or calorimetric assays are particularly convenient. A method that allows for flexibility can be additionally valuable, such as a scheme that can be used at any point in the development of a transgenic variety. A most preferred method would allow selection in culture, identification in breeding and introgression activities, as well as identification and discrimination at the field level.
Concerns Associated with Field Release of Transgenic Plants
It has been suggested that the release of genetically modified crops could lead to environmental damage because of their expression of genetic potential that would not ordinarily be attained by natural selection or via sexual recombination. It has further been suggested that released transgenic plants could invade natural ecosystems either through the spread of the plants themselves or through hybridization with wild relatives. These issues have been extensively debated and experimentation has been initiated to test for continued survival of transgenic plants and transfer of traits from crop species to wild relatives. (e.g. University of California, Risk assessment in agricultural biology: proceedings of an international conference, 1990, Casper, R., & Landsman, J., 1992; The bio-safety results of field tests of genetically modified plants and microorganisms. Proceedings of the 2nd International Symposium on The Biosafety Results of Field Tests of Genetically Modified Plants and Microorganisms, 1992, Goslar, Germany, Dale, P. et al., 1992; The field release of transgenic plants, The British Crop Protection Council. Brighton Crop Protection Conference: Pests and Diseases , Vols. I, II and III; Proceedings of the 3rd International Symposium on The BioSafety Results of Field Tests of Genetically Modified Plants and Microorganisms, 1994, Monterey, Calif., Jones, D. D., 1994)
The consensus of the studies and experimental results achieved to date supports the view that the degree of potential spread of transgenes to wild relatives is highly dependent upon the species and environmental conditions. Crossing with relatives is not likely with some species and probable for others (Raybould & Grey, J., Applied Ecology 30: 199-219, 1993). The degree to which any transformed plant can be invasive of other habitats, and hence the environmental risk, is also dependent on the plant species itself. Many crops are highly specialized and adapted to non-competitive cultivation practices and, thus, they are not generally considered a serious environmental risk (Dale et al., Plant Breeding 111:1-22, 1993; Fishlock, D., The PROSAMO Report, published by the Laboratory of the Government Chemist, Queens Road, Teddington, Middlesex, UK TW11 OLY).
However, it is generally agreed that there are probably some risks that certain crop plants, ornamentals or plants cultivated for natural pharmacological purposes could become weedy pests since many of the weedy species currently affecting agricultural production were at one time introduced from another environment, frequently for ornamental, culinary or medicinal reasons (Keeler, K. H., Biotechnology 7: 1134-1139, 1989).
While years of study will be required to understand fully the potential impact of transgenic plants on the environment, a potentially more serious near term problem relates to contamination of agricultural production with novel traits from transgenic plants. Both Xenia effects (direct effect of cross pollen on the composition of seed) and volunteers or seed that remain in the field can contaminate subsequent agricultural production. Although such events have not been a major problem in the past, the inadvertent contamination of crops intended for general consumption with visually indistinguishable varieties developed for other purposes, for example, crops that contain a pharmaceutically active protein, has become an issue of particular concern. Accordingly, the ability to discriminate transgenic crops in a simple and reliable way is of value.
Currently, physical isolation combined with border rows that function as pollen traps have been employed to contain plants with transgenic traits under study and development (e.g., Agriculture and Agri-Food Canada, Regulatory Directive 94-08; Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits, Regulatory Directive 94-09: The Biology of Brassica napus L. (Canola/Rapeseed), Regulatory Directive 95-01; Field Testing Plants with Novel Traits in Canada). However, with increasing commercial production of transgenic plants, the potential for contamination within a commodity increases dramatically. This potential contamination has become a major concern for the oilseed rape industry and will become a significant issue for other major crops (e.g. corn), as greater numbers of different recombinant genotypes reach the market place.
Contamination of commercial crop production with traits from another cultivar that affect quality and performance is a potentially serious problem. However, because of possible contamination of food products, a potentially more serious problem is the use of canola (or other crops) as a production vehicle for heterologous proteins of commercial or medicinal value. Although production standards as set out above can be implemented to preserve the identity of individual transgenic lines and reduce unintended contamination, the outflow of genes to other cultivars may eventually occur. The issues relating to the occurrence and spread of genes that do not impart a distinctive morphology or an easily identifiable trait (such as herbicide tolerance) have not yet been resolved.
Though many techniques have been used to introduce genes into plants, genetic constructs in the prior art do not include features that are useful for identifying and selecting transgenic plant cells in culture or for controlling the persistence or potential spread of the transgenes. Accordingly, genetic constructs that operate within a mechanism that permits discrimination of transgenic plants from non-transgenic plants, or discrimination among transgenic plants carrying different traits, would solve the contamination problem. A mechanism that has no effect on non-transgenic plants, yet allows the plants containing a specific transgene to be eliminated as they are identified, would also provide a solution. Furthermore, a mechanism which selectively removes crop plants containing specific transgenes from other commercial crops not having those transgenes would also be valuable.
Methods for Transformation of Plants
In general, two methods for introducing DNA into plant cells are currently in widespread use. The first involves the use of Agrobacterium, or similar soil bacteria, to transfer DNA. Target plant tissues or cells are co-cultivated with a suitable Agrobacterium strain that injects plasmid DNA into plant cells (Schilperoot, R. A. et al., U.S. Pat. No. 4,940,838; Schilperoot, R. A. and Hoekema, A., U.S. Pat. No. 5,464,763). Subsequently, the individual transformed plant cells are regenerated into whole plants.
The Agrobacterium transformation system is based historically on the use of a natural bacterial vector that is the causal agent of crown gall disease. Crown gall disease represents the results of a natural form of plant transformation. The naturally occurring tumor inducing (Ti) plasmids of Agrobacteria comprise: the DNA sequences needed for the transfer of DNA into plant cells; the DNA sequences needed for integration of foreign DNA into the host plant DNA, called the border fragments; and genes, called oncogenes, that result in the formation of plant growth regulatory substances that cause the formation of galls at the site of infection. The Ti plasmids also typically code for genes that result in the formation of certain types of unusual amino acids (opines) that can be metabolized by Agrobacteria, but not by plant cells. In the natural system, the portion of the Ti plasmid which is transferred to the recipient plant host (the T-DNA) usually contains all these genes and DNA sequences.
The oncogenes of the tumorigenic Agrobacterium strains have been extensively studied. Generally, there are two types of oncogenes on the Agrobacterium plasmid: the tmr oncogene and the tms oncogenes. The tmr oncogene (also known as the ipt gene) encodes an enzyme which synthesizes isopentyl-adenosine 5′-monophosphate which is a cytokinin plant hormone that induces shoot formation in a suitable host. The oncogenes referred to as tms (comprising tms oncogene 1 and tms oncogene 2) encode enzymes responsible for auxin overproduction in suitable hosts, leading to the production of roots. When combined, the tms and tmr genes usually lead to the production of crown galls on suitable hosts.
Plants which contain the Ti oncogenes are phenotypically abnormal, having crown gall tumors or curled and twisted leafs due to growth hormone imbalance. These abnormal plants are unsuitable for commercial applications. Although these plants can be easily identified following transformation, they do not form morphologically normal plants. Accordingly, the Agrobacterium Ti plasmid has been modified in a variety of ways, and typically by removal of the oncogenes, to become a tool for the introduction of DNA into plant cells. Generally, Agrobacterium transformation methods that have been used to date have used Ti plasmids in which the genes that result in the formation of cytokinins and auxins and the genes for opine synthesis have been removed. Such plasmids are generally referred to as being “disarmed”. Accordingly, an “armed” Ti plasmid is generally considered to contain an oncogene.
The Ti plasmid for use in plant transformation has further been engineered to contain restriction sites, for the convenient introduction of foreign genes between or adjacent to one or more of the border fragments, and genes for identification and selection of transformed cells, such as antibiotic resistance genes or β-glucuronidase synthase (GUS) genes. Replication genes are often introduced to the Ti plasmid to allow replication of the plasmid in non-Agrobacterial hosts. The vir (virulence) genes that are required for the mobilization of DNA and transfer of the plasmid from the bacterial cell are often retained on a separate plasmid or in a location of the Ti plasmid distinct from that containing the DNA which is to be transferred and, as such, are not transferred to the recipient plant cells.
The second wide spread technology employed to generate transformed plants involves the use of targeted microprojectiles. These methods have been employed to transform both monocots and dicots that are recalcitrant to Agrobacterium methods. A variety of different microprojectiles and methods of bombardment have been described in, for example, Sanford, et al., U.S. Pat. No. 4,945,050; McCabe, et al., U.S. Pat. No. 5,149,655; Fitzpatrick-McElligott et al., U.S. Pat. No. 5,466,587; and Coffee et al., U.S. Pat. Nos. 5,302,523, 5,464,765. The DNA introduced using targeted microprojectiles comprises similar functional features for expression in plant cells, equivalent to those introduced via Agrobacterium systems, for example, the vector used often comprises engineered sites for foreign gene insertion and genes needed for identification or selection of transformants.
Other methods that have been used to obtain transformed plants include: microinjection directly into cell nuclei (Crossway et al., U.S. Pat. No. 4,743,548); and direct DNA uptake by protoplasts (Paszkowski et al., U.S. Pat. Nos. 5,231,019, 5,453,367)
Although the general approach to plant transformation is well-understood, the practical application of plant transformation processes is often limited by genotypic response of plant cells to transformation and culture conditions. It is not unusual for only one or two narrow genotypes within a species to be amenable to transformation. Accordingly it is not simple, or in some cases possible, to efficiently transform all genotypes within a crop species. Despite numerous attempts to alter culture and transformation protocols, some plant genotypes are recalcitrant to transformation by techniques that are efficient within other genotypes. Thus in many cases a specific genotype that is amenable to transformation is first used, then the transgenic event is crossed, or “introgressed” into germplasm that is recalcitrant to the same transformation method. Although this method may allow for the eventual introduction of a transgene into a line that can not be transformed without undue effort, the process takes time and effort since one has to select for the transgene at every sexual cross.
Thus a method that allows rapid discrimination of plants into which the transgene has been introduced by either transformation or introgression, would greatly facilitate production of transgenic plants. In particular, a non-destructive visual assay would allow rapid screening of large numbers of breeding lines and segregating populations. This screening process, if applied at the seedling stage or even at the stage of seed development (e.g. embryo rescue), would find utility within commercial varietal production programs by allowing selection of lines at an early stage, thus eliminating the need to grow plants to maturity, thereby saving time and land resources. Such a method could also be used to eliminate significant number of null lines from cultivation and allow for a streamlined breeding and introgression process. In particular, such a method would be useful for producing Brassica plants carrying transgenes for input or output traits, including high value output traits.
Brassica Transformation
Transformation of members of the Cruciferae family by Agrobacterium and other methods has been reported. However, many of the reports that relate specifically to Brassica transformation have detailed the difficulty in routinely obtaining transformed Brassica species by Agrobacterium mediated transformation. Many of the reports have shown success with one or two particular varieties, but there is no teaching of detailed methods that are generally applicable to all species within the Brassica genus. Although many manipulations of culture conditions can be employed, some varieties have proven to be extremely difficult to transform by previously reported methods. Thus significant effort is expended for the introduction of transgenes in these genotypes by crossing and introgression. Any improvements and discoveries that allow for the reliable generation of transgenic plants from these genotypes or allow for a more efficient means to introgress these traits would be a significant improvement over the art.
Many of the initial Brassica transformation studies were carried out with a single genotype, B. napus cv Westar (Radke et al, Theor. Appl. Genet. 75:685-694, 1988; Moloney et al., Plant Cell Reports 8:238-242, 1989; Moloney et al., 1989, U.S. Pat. No. 5,188,958; Moloney et al., 1989, U.S. Pat. No. 5,463,174). Westar was a convenient choice since it responded to tissue culture and transformation protocols described in the references cited above and allowed recovery of transgenic plants. Westar remains the genotype of choice for transformation experiments; however, the agronomic properties of the variety are considered poor by comparison with recent cultivars. Hence a gap remains in reliable transformation technology and commercial genotypes of Brassica napus oilseed.
Moreover, many Brassica transformation studies conducted using the described methods, or variations thereof, have produced results that are highly variable and are dependent upon the innate response of the specific plant materials to the transformation protocol. As an example, the transformation frequencies that have been achieved for Brassica napus are sometimes variable and very low (Fry et al., Plant Cell Reports 6:321-325, 1987; Mehra-Palta et al., In Proc 8th Int. Rapeseed Congress, Saskatoon, Saskatchewan, 1991; Swanson and Erickson, Theor. Appl. Genet. 78:831-835, 1989). Variable and often low transformation frequencies have also been observed with other Brassica species, such as B. oleracea (Christie and Earle, In Proc 5th Crucifer Genetics Workshop, Davis, pp 46-47, 1989; Metz et al., Plant Cell Reports 15: 287-292, 1995; Eimert and Siegemund, Plant Molec. Biol. 19:485-490, 1992; DeBlock et al., Plant Physiol. 91:694-701, 1989; Berthomieu and Jouanin, Plant Cell Reports 11:334-338; Toriyama et al, Theor. Appl. Genet. 81:769-776, 1991); B. rapa (Radke et al., Plant Cell Reports 11:499-505, 1992; Mukhopadhyay et al, Plant Cell Reports 11:506-513, 1992); B. juncea (Barfield and Pua, Plant Cell Reports 10:308-314, 1991; Deepak et al., Plant Cell Reports 12:462-467, 1993; Pua and Lee, Planta 196:69-76, 1995); B. nigra (Gupta et al, Plant Cell Reports 12:418-421, 1993); and B. carinata (Narasimhulu et al., Plant Cell Reports 11:359-362, 1992; Babic, M. Sc. Thesis, Univ of Saskatchewan, 1994).
The many Brassica species, varieties and cultivars represent a very diverse group with radically different morphologies and physiological characteristics. Many Brassica species of commercial interest do not respond well or at all to the methods previously described. In particular, Brassica napus oilseed species with unusual fatty acid compositions appear to be recalcitrant to conventional transformation efforts. One genotype, typified by the variety AG019, as described in U.S. Pat. No. 5,965,755 is a high oleic acid, low linoleic acid genotype that is unresponsive to conventional transformation methods, for example, that described by Moloney (ibid). The variety AG019 and varieties derived therefrom have valuable fatty acid compositions that provide oil with improved oxidative stability and nutritional value. Crossing with a conventional oil profile Brassica napus, for example, a transformed Westar, as a means to introduce a transgene, causes a loss in oil profile and requires significant breeding efforts to reconstruct the desired oil profile. In most cases it is uncertain that this can be attained. Thus, a transformation method is required which is useful for transformation of recalcitrant Brassica, particularly Brassica napus oilseed species with altered oil profiles, more particularly transformation of Brassica species AG019 and progeny derived therefrom.
Accordingly, methods to transform recalcitrant genotypes of Brassica will be valuable. Furthermore, methods to identify transformed plant cells, and plants and progeny derived therefrom will also be valuable particularly if the methods are simple, non-destructive in nature and allow visual identification of the plants or cells that contain the transgene of interest. If such methods further provide discrimination at the field level, then a wide range of appliations is feasible.
Methods have been developed that address these needs to some degree. Visual marker genes such as the β-Glucuronidase or GUS gene are available, but require a biochemical or histochemical assay. Genes which respond to applied chemicals are typified by so-called “conditionally lethal” genes. However, these typically lead to a lethal phenotype, hence are not useful for tracking transgenes in a breeding process. Accordingly, it is an object of the present invention to provide a conditionally lethal gene that is useful in a breeding program, as well as other commercially important objectives.
Conditionally Lethal Genes
Conditionally lethal traits and genes which impart these traits, are known. Many conditionally lethal genes lead to a lethal phenotype and plant death. However, some conditionally lethal genes can be used in a fashion that will not necessarily lead to cellular death. An example of such a conditionally lethal gene is the Agrobacterium Ti plasmid tms oncogene 2. This oncogene codes for the enzyme indoleacetamide hydrolase (IAMH) that, in combination with Agrobacterium oncogene 1 which codes for indoleacetamide synthase (IAMS), forms part of the indoleacetic acid (IAA) synthesis pathway typical of this type of bacterium.
The formation of indoleacetic acid by plants takes place by a different pathway from that of Agrobacterium. Hence, the expression of IAMH (oncogene 2) in plants does not result in the formation of indoleacetic acid because the substrate for the enzyme, indoleacetamide, is not present in plant cells. However, application of indoleacetamide to plants expressing the IAMH gene results in the rapid accumulation of IAA. Even though IAA is a naturally occurring auxin plant growth regulator, uncontrolled high levels of IAA rapidly disturb cellular metabolism resulting in senescence and cell death. The enzyme IAMH is capable of hydrolyzing other indoleamide-related substrates including naphthalene acetamide resulting in production of the well known synthetic plant growth regulator naphthalene acetic acid (NAA).
The use of a conditionally lethal gene, such as the IAMH oncogene, for roguing maize plants is described in U.S. Pat. No. 5,180,873 (Jorgensen) issued January, 1994. Jorgensen teaches transformation of plants to contain a conditionally lethal gene. The plants are subsequently subjected to linkage analysis, then selected for close linkage between the lethal gene and a target locus which is either pre-existing or introduced by traditional breeding techniques. U.S. Pat. No. 5,426,041 of Fabijanski et al issued June 1995, teaches a method of hybrid seed production using IAMS in combination with IAMH. Neither patent teaches a method for using the oncogene 2 in a non-lethal means or for selection during and after transformation. Neither patent teaches a method for using oncogene 2 for selectively removing related species having acquired the transformed genetic material.
Thus a conditionally lethal gene within a genetic construct also containing a novel trait offers a convenient means to control spread of the novel trait. However, because the lethal gene does not provide a non-destructive means to identify or select transformed plant cells, specifically Brassica cells, traditional conditionally lethal genes do not solve the present problem of selecting, identifying and tracking transgenic plants and progeny derived therefrom.
Accordingly, another object of the present invention is to modify and use a previously identified conditionally lethal gene to track transgenes during the breeding and commercialization process as well as under field conditions.